The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation (original) (raw)

1. Mathias Stotz1,8,
2. Saadia Qamar2,3,
3. Katharina Kramer2,3,
4. Janosch Hennig4,5,
5. Thomas Schubert6,
6. Patrick Löffler7,
7. Gernot Längst6,
8. Rainer Merkl7,
9. Henning Urlaub2,3 and
10. Gunter Meister1,9
11. 1Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
12. 2Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
13. 3Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, 37075 Göttingen, Germany;
14. 4Group Biomolecular NMR, Institute of Structural Biology, Helmholtz Zentrum München-German Research Center for Environmental Health, 85764 Neuherberg, Germany;
15. 5Center for Integrated Protein Science Munich, Biomolecular NMR, Department Chemie, Technische Universität München, 85748 Garching, Germany;
16. 6Institute for Biochemistry III, University of Regensburg, 93053 Regensburg, Germany;
17. 7Institute of Biophysics and Physical Biochemistry, University of Regensburg, 93053 Regensburg, Germany
18. ↵8 These authors contributed equally to this work.

Abstract

The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein–protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed β propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

• BRAT
• RNA binding
• TRIM-NHL
• gene regulation
• hunchback
• translational repression

Footnotes

• ↵9 Corresponding authors
  E-mail gunter.meister{at}vkl.uni-regensburg.de
  E-mail inga.loedige{at}vkl.uni-regensburg.de

• Supplemental material is available for this article.

• Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.236513.113.

• Received December 16, 2013.

• Accepted February 20, 2014.

• © 2014 Loedige et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.